During FY2010, we extended studies of HIV Env-receptor interactions and neutralization mechanisms, with emphasis on features that distinguish the functional Env trimer from the more highly studied gp120 monomer. We also continue to develop anti-HIV agents based on the molecules involved in entry. 1) Epitope masking within the HIV-1 Env trimer. We analyzed the epitope masking mechanism of masking for an unusual mAb D19. The epitope, on the V3 loop, is fully exposed on monomeric gp120;within the functional Env trimer, the epitope is in R5 strains (rendering the resistant to neutralization), but available in CXCR4-using strains (which are neutralization sensitive. Deletion of the V1V2 loop exposes the D19 epitope on R5 trimers. We employed a previously developed functional complementation system to address the question of whether V1V2 masking of the V3 eptiope occurs by mechanism that is cis (intrasubunit, i.e. V1V2 masking of V3 within the same gp120 protomer) or trans (intersubunit, i.e. V1V2 masking of V3 within adjacent gp120 protomers). In 2010 by examining complementation and neutralization with various combinations of co-expressed Env constructs (mutations, deletions, etc.) we obtained clear evidence for a cis masking mechanism. These functional results support one of the two alternate mechanisms for arrangement of the variable loops within the Env trimer that have been proposed based on structural and modeling studies. 2) 2-phases of the CD4-gp120 interaction. We have identified a region of CD4 that may be involved in induction/stabilization of the coreceptor binding site on gp120, rather than in binding to gp120 per se. In 2010 we found that specific mutations in this region have only modest effects on binding of soluble CD4 to monomeric gp120, but appear to abolish its binding to the functional trimer, as assessed by its inability to neutralize infectivity or to induce Env-mediated fusion with target cells expressing CCR5 but not CD4. Efforts were initiated to probe these actions in molecular detail, including collaborative studies to employ single molecule FRET analyses. 3) Producer cell-type distinctions in HIV virion sensitivity to carbohydrate-binding neutralizing protein. Based initially on our studies of the bifunctional neutralizing protein sCD4-17b, we expanded on our previous confirmation of findings that HIV virions produced by PBMC are much less sensitive than isogenic virions produced by a cell line to neutralization by the glycan-recognizing 2G12 mAb. Preliminary experiments in 2010 demonstrate similar distinctions in neutralization sensitivity by the carbohydrate binding proteins cyanovirin-N and griffithsin, although the effects are strain-dependent. These results raise important questions about the potential antiviral utility of these lectins, which are being actively pursued as candidate topical microbicides. 4) Anti-HIV immunotoxins. Preliminary collaborative studies in the SIV/rhesus macaque model in 2010 have provided suggestive evidence that CD4-PE40 may have some efficacy in depleting infected cells that persist in the face of suppressive antiretroviral therapy. A 2010 pre-IND meeting with the FDA has provided important guidelines for further development of 3B3-PE38 toward a Phase 1 trial to test this in the context of viral suppression by HAART. We are also designing new assays to test the efficacy of 3B3-PE38 against virus obtained from HAART-suppressed persons;this will be a critical component in selection of candidate subjects for future clinical trials.